All living creatures, be it man or the smallest bacteria have one function in common known as respiration. During respiration, two important functions are performed in living things. In the first, electrons that were generated during catabolism are disposed of and in the second, ATP (also known as adenosine tri-phosphate) is produced to provide energy for the cell.
There are two types of respiration: (i) aerobic respiration and (ii) anaerobic respiration. Aerobic respiration requires oxygen, but oxygen is not required for anaerobic respiration, often called “fermentation” in bacteria. Instead, other less-oxidizing substances such as sulfate (SO42−), nitrate (NO3−), sulfur (S), or fumarate are used. These terminal electron acceptors have smaller reduction potentials than O2, meaning that less energy is released per oxidized molecule. Anaerobic fermentation is, therefore, energetically less efficient than aerobic respiration. Nonetheless, it has value and allows the cells to continue living even with no or reduced O2.
Anaerobic fermentation and aerobic respiration have been the two metabolic modes of interest for the industrial production of chemicals from microbes such as E. coli, Lactobacillus and yeast. Oxygen rich respiration offers very efficient cell growth (growth rate and yield) and converts a high percentage of the carbon source into carbon dioxide and cell mass (see Table 1). Anaerobic fermentation, on the other hand, results in poor cell growth and the synthesis of several fermentation products at high yields (e.g. lactate, formate, ethanol, acetate, succinate, etc.).
However, producing chemicals via oxygen rich processes costs much more than using anaerobic methods for two reasons. First, aerobic fermenters are more expensive to build, due to both the higher cost per unit and the need for smaller fermenters with reduced economy of scale. Secondly, the aerobic fermenters are more costly to operate than their anaerobic counterparts due to low solubility of oxygen, which in turn requires high energy input to ensure appropriate supply of oxygen to the cells. This is especially relevant for the production of commodity chemicals, where fermentation costs can represent 50-90% of the total production cost.
TABLE 1RESPIRATORY VS FERMENTATIVE METABOLISMAnaerobicAnaerobicAerobicVariableFermentationRespirationRespirationGrowth RateLOWIntermediateHIGHCell MassLOWIntermediateHIGHProduct YieldsHIGHHigh/IntermediateLOWCapital CostLOWLOWHIGHEnergy InputLOWLOWHIGH
Therefore, anaerobic methods are usually preferred where possible, and it is typical to grow cells to a large number aerobically, and then switch the cells to anaerobic culture for the production of desired chemicals. Often, however, the method is less than completely successful, resulting in poor yields and rates.
US20100317086 describes a three-stage process, where the bacteria are first acclimated to low O2 conditions before switching to anaerobic conditions, and this helps to improve yields of various biological chemicals.
However, there is still a need to maximize chemical production, while maintaining robust cell growth, and optimizing production yields. One way of optimizing yield is to directly attempt to increase the genes resulting in the desired product. Another method, would be to directly downregulate a competitive pathway. However, direct methods have limitations, can be difficult to fine tune, and are often not satisfactory. We introduce herein indirect methods of influencing flux, which are amenable to fine tuning.